Results

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ResultsmiRNA and small RNA expression analysesEarlier studies showing the presence of miR-21 and miR-155 in the serum/plasma of patients with cancer [25,26] prompted us to evaluate their levels in the sera of healthy subjects and patients with breast cancer. U6, 5S, miR-16, RNU66, RNU49, RNU19, and SNORD44 levels were also analyzed in these samples to identify a small RNA expressed at a similar level in equal volume of sera from both healthy subjects and patients with cancer to serve as a normalization control. Among these, miR-16 has previously been used as a normalization control for serum miRNA profiling studies [25]. RNU66, RNU48, and RNU19 were undetectable. miR-16 is one of the most abundant miRNAs in the serum (average cycle threshold (CT) of 24) compared with any other RNA analyzed, and the abundance of this miRNA in the serum was similar between healthy subjects and patients with cancer (Table 4 and Figure 1a)...

Serum U6 and 5S in relation to ER/PR and nodal status of primary tumorsWe next determined whether the upregulation of U6 and 5S observed above is unique to specific subtypes of breast cancer. ER/PR status of 36 tumors, out of 39 patient samples used in the study, was known; 24 and 12 were ER/PR+ and ER/PR-, respectively. In this subgroup analysis, U6, but not 5S, levels were higher in the sera of patients with ER/PR+ breast cancer compared with healthy subjects with or without normalization with miR-16 and SNORD44 (Table 5 and Figure 3). Despite a small sample size, sera of patients with ER/PR- demonstrated elevated U6 and 5S levels compared with healthy subjects when miR-16 or SNORD44 was used as an internal control (Table 5 and Figure 3). miR-21 level was similar between healthy subjects and patients with cancer of either subtype. Note that the differences in the levels of these RNAs between ER/PR+ and ER/PR- were not statistically significant (data not shown)...

U6 levels are elevated in sera of patients with breast cancer, irrespective of metastasisWe collected sera from 15 healthy subjects, 15 clinically disease-free patients with breast cancer, and 15 patients with stage IV metastatic disease. RNA from the three groups (cohort 2) was prepared at the same time from an equal volume of sera. The cDNA was prepared with 25 ng of RNA and analyzed for the levels of U6, 5S, miR-21, and miR-16. In this cohort, as with the first cohort, U6 level was significantly elevated in the sera of clinically disease-free patients with breast cancer without normalization (Table 6 and Figure 4) (5.35-fold, P = 3.09 x 10-5) or with normalization with SNORD44 (6.6-fold, P = 0.00028)...

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